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CAPACITATIVE Ca2+entry involves the influx of Ca2+across the sarcolemma in response to depletion of intracellular Ca2+stores. 1–4 Capacitative Ca2+entry is insensitive to voltage-gated Ca2+channel inhibitors. 2,3 The mechanisms involved in regulating capacitative Ca2+entry are not well understood. We recently demonstrated that capacitative Ca2+entry exists in canine pulmonary artery smooth muscle cells (PASMCs) and serves to refill the sarcoplasmic reticulum Ca2+pool. 5 Moreover, we observed that capacitative Ca2+entry is required to maintain α-agonist–induced oscillations in intracellular Ca2+concentration ([Ca2+]i) and is involved in the contractile response to α-adrenoreceptor activation. 5 Because capacitative Ca2+entry is involved in the regulation of [Ca2+]iand vasomotor tone, it could serve as a cellular target for anesthetic agents that exert vasoactive effects.

The goal of the current study was to investigate the effect of propofol on capacitative Ca2+entry in PASMCs. We tested the hypothesis that propofol would attenuate capacitative Ca2+entry in PASMCs. The rationale for this hypothesis is based on two factors. First, propofol is known to inhibit voltage-gated influx of extracellular Ca2+in vascular smooth muscle. 6–8 Second, we have demonstrated that intravenous anesthetics, 9,10 including supraclinical concentrations of propofol, 9 attenuate phenylephrine-induced [Ca2+]ioscillations in PASMCs, which require capacitative Ca2+entry. Because a tyrosine kinase is involved in the signal transduction pathway for capacitative Ca2+entry in PASMCs, 5 we also tested the hypothesis that tyrosine kinase inhibition would prevent the effects of propofol on capacitative Ca2+entry. Finally, we investigated the effects of protein kinase C (PKC) activation and inhibition on capacitative Ca2+entry and assessed the extent to which PKC is involved in propofol-induced changes in capacitative Ca2+entry.

Materials and Methods

Animals

Pulmonary arteries were isolated from adult mongrel dogs. The technique of euthanasia was approved by the Cleveland Clinic Institutional Animal Care and Use Committee (Cleveland, OH). All steps were performed aseptically during general anesthesia with intravenous pentobarbital sodium (30 mg/kg) and intravenous fentanyl citrate (20 μg/kg). The dogs were intubated and ventilated, exsanguinated by controlled hemorrhage via
a femoral artery catheter, and euthanized with electrically induced ventricular fibrillation. A left lateral thoracotomy was performed, and the heart and lungs were removed en bloc
. The pulmonary arteries were isolated and dissected in a laminar flow hood during sterile conditions.

Cell Culture of Pulmonary Artery Smooth Muscle Cells

Primary cultures of PASMCs were obtained as previously described. 11 Intralobar pulmonary arteries (ID = 2–4 mm) were carefully dissected and prepared for tissue culture. Explant cultures were prepared according to the method of Campbell and Campbell, 12 with minor modifications. Briefly, the endothelium and adventitia were removed together with the most superficial part of the tunica media. The media was cut into 2-mm2pieces and explanted in 25-cm2culture dishes nourished by D-MEM/F-12 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum and a 1% antibiotic–antimycotic mixture solution (10,000 units/ml penicillin, 10,000 μg/ml streptomycin, 25 μg/ml amphotericin B) and kept in a humidified atmosphere of 5% CO2:95% air at 37°C. PASMCs began to proliferate from explants after 7 days in culture. Cells were allowed to grow for an additional 7–10 days before being subcultured nonenzymatically to 35-mm glass dishes designed for fluorescence microscopy (ΔT system; Bioptechs Inc., Butler, PA). Cells were used for experimentation within 72 h. Cells from the first and second passage were used for experiments. More than 90% of the cells stained positive for smooth muscle α actin. 11

Intracellular Ca2+concentration was measured as previously described. 11 Culture dishes containing fura 2–loaded PASMCs were placed in a temperature-regulated (37°C) chamber (Bioptechs, Inc.) mounted on the stage of an Olympus IX-70 inverted fluorescence microscope (Olympus America Inc., Lake Success, NY). Fluorescence measurements were obtained from either individual PASMCs or from a cluster (two to three cells) of neighboring cells in a culture monolayer using a dual-wavelength spectrofluorometer (Deltascan RFK6002; Photon Technology International, Lawrenceville, NJ) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The volume of the chamber was 1.5 ml. The cells were superfused continuously at 1 ml/min with Krebs-Ringer buffer, which contained 125 mm NaCl, 5 mm KCl, 1.2 mm MgSO2, 11 mm glucose, 2.5 mm CaCl2, and 25 mm HEPES, at pH 7.40 adjusted with NaOH. The temperature of all solutions was maintained at 37°C in a water bath. Solution changes were accomplished rapidly by aspirating the buffer in the dish and transiently increasing the flow rate to 10 ml/min. Just before data acquisition, background fluorescence (i.e.
, fluorescence between cells) was measured and subtracted automatically from the subsequent experimental measurements. Fura 2 fluorescence signals (340, 380, and 340/380 ratio) originating from PASMCs were continuously monitored at a sampling frequency of 25 Hz and were collected using a software package from Photon Technology International.

Propofol, thapsigargin, phorbol 12-myristate 13-acetate (all obtained from Research Biochemical International, Natick, MA), tyrphostin 23 (Calbiochem, La Jolla, CA), and bisindolylmaleimide 1 (Sigma, St. Louis, MO) were all dissolved in dimethyl sulfoxide as stock solutions. Aliquots of each stock solution were diluted 1:1000 in Krebs-Ringer buffer to achieve final concentrations in the bath. Similar dilutions of dimethyl sulfoxide in Krebs-Ringer buffer have no effect on [Ca2+]i. Pure propofol was used to avoid any effects of the intralipid emulsion on the fluorescence signal.

Data Analysis

Peak and sustained increases in [Ca2+]iwere measured in PASMCs when the superfusion solution was switched from a Ca2+-free solution to a solution containing 2.2 mm Ca2+. Peak and sustained fluorescence ratio values were averaged before and after each intervention and are expressed as percent of control. The control response to which all interventions were compared was the first capacitative Ca2+entry response after thapsigargin pretreatment. This value was set at 100%. Therefore, each cell served as its own control. The “peak” response was calculated as the fluorescence change from baseline to peak fluorescence. The “sustained” response represents the fluorescence values measured 5 min after reintroduction of Ca2+to the buffer. Results are presented as mean ± SEM. Statistical analysis was performed using repeated-measures analysis of variance followed by Bonferroni–Dunn post hoc
testing. Differences were considered statistically significant at P
< 0.05.

Results

Capacitative Ca2+Entry in Pulmonary Artery Smooth Muscle Cells

Capacitative Ca2+entry can be triggered by thapsigargin-induced depletion of intracellular Ca2+stores. In the absence of extracellular Ca2+, thapsigargin (1 μm) increased [Ca2+]iby 182 ± 11%, followed by a return of [Ca2+]ito baseline values (fig. 1, top). Once the baseline fluorescence signal had stabilized, the extracellular Ca2+concentration ([Ca2+]o) was restored (2.2 mm) in the continued presence of thapsigargin (fig. 1, top). Restoring [Ca2+]oresulted in a rapid peak increase (246 ± 12% of baseline) in [Ca2+]iand a sustained increase (187 ± 7% of baseline) in [Ca2+]i(i.e.
, capacitative Ca2+entry was induced). The sustained increase in [Ca2+]ireturned to baseline when [Ca2+]owas removed. The reproducibility of inducing capacitative Ca2+entry was assessed by sequentially removing and restoring [Ca2+]othree consecutive times in the continued presence of thapsigargin. There were no significant differences in the peak or sustained increases in [Ca2+]iwhen [Ca2+]owas restored three consecutive times (fig. 1, bottom).

After depletion of sarcoplasmic reticulum Ca2+stores with thapsigargin, capacitative Ca2+entry was compared in the absence or presence of propofol, which was added to the superfusion buffer 5 min before restoring [Ca2+]oa second time (fig. 2, top). Propofol had no effect on baseline [Ca2+]ibefore the addition of [Ca2+]o. Propofol caused dose-dependent decreases in both the peak and sustained increases in [Ca2+]iwhen [Ca2+]owas restored (fig. 2, bottom). After washout of propofol, capacitative Ca2+entry was similar in magnitude to the response measured before propofol administration (fig. 2, top).

Role of Tyrosine Kinases in Propofol-induced Attenuation of Capacitative Ca2+Entry

We previously demonstrated that tyrosine kinases play a role in regulating capacitative Ca2+entry in PASMCs. 5 In the current study, tyrphostin 23 (100 μm) was used to inhibit tyrosine kinases. Tyrosine kinase inhibition attenuated both the peak (67 ± 4% of control) and sustained (75 ± 5% of control) increases in [Ca2+]imediated through capacitative Ca2+entry (fig. 3). However, in the presence of tyrosine kinase inhibition, propofol (100 μm) further attenuated the peak (46 ± 4% of control) and sustained (55 ± 2% of control) increases in [Ca2+]iwhen [Ca2+]owas restored (fig. 4).

Role of Protein Kinase C in Propofol-induced Attenuation of Capacitative Ca2+Entry

Protein kinase C has been implicated in the regulation of capacitative Ca2+entry in a variety of cell types. 14–16 However, the extent to which PKC is involved in capacitative Ca2+entry in PASMCs has not been previously investigated. Activation of PKC with phorbol 12-myristate 13-acetate (1 μm) attenuated both the peak (48 ± 1% of control) and sustained (53 ± 3% of control) increases in [Ca2+]imediated via
capacitative Ca2+entry (fig. 5). In contrast, PKC inhibition with bisindolylmaleimide (1 μm) potentiated both the peak (132 ± 11% of control) and sustained (120 ± 4% of control) increases in [Ca2+]iwhen [Ca2+]owas restored (fig. 6). Moreover, in the presence of PKC inhibition, propofol (100 μm) had no effect on capacitative Ca2+entry (fig. 7). Thus, PKC inhibition abolished the propofol-induced attenuation in capacitative Ca2+entry.

It is well known that [Ca2+]iis an important determinant in the regulation of cardiac and smooth muscle contraction. In vascular smooth muscle, agonist-induced increases in [Ca2+]iprimarily occur via
release of Ca2+from intracellular stores by a 1,4,5 inositol triphosphate–dependent mechanism. In addition, some agonists can trigger Ca2+influx across the sarcolemma, primarily via
voltage-gated or receptor-operated Ca2+channels. An increase in [Ca2+]iactivates the myosin light chain kinase through a calmodulin-dependent mechanism, resulting in phosphorylation of the myosin light chains and initiation of contraction. 17 [Ca2+]iis ultimately restored either by pumping the Ca2+out of the cell via
the sarcolemmal Ca2+ATPase or Na+–Ca2+exchanger, or by resequestering Ca2+into the intracellular store by the sarcoplasmic reticulum Ca2+ATPase. 18,19

In addition to the aforementioned sarcolemmal ion channels, Ca2+influx can also be controlled by the filling-state of the intracellular Ca2+store. The concept of capacitative Ca2+entry was first postulated by Putney. 1 According to his model, depletion of intracellular Ca2+stores results in activation of a Ca2+influx pathway that is somehow sensitive to the state of filling of intracellular Ca2+stores. Influx of Ca2+via
capacitative Ca2+entry refills the intracellular Ca2+stores that have been depleted in response to agonist activation. In the current study, thapsigargin was used as a tool to deplete the sarcoplasmic reticulum pool of Ca2+in the absence of extracellular Ca2+and thereby activate capacitative Ca2+entry. Consistent with our previous study, 5 the amplitude of the thapsigargin-induced increase in [Ca2+]iwas variable, which likely reflects differences in the size of the sarcoplasmic reticulum Ca2+store in different cells. The size of the sarcoplasmic reticulum Ca2+store may depend on the cell passage number, the phase of the cell cycle, the length of time in serum-free medium, or whether the response was derived from an individual cell or a cluster of two to three neighboring cells. Restoring [Ca2+]ostimulated capacitative Ca2+entry, which was typically characterized by both a peak and sustained increase in [Ca2+]i. The rapid peak increase in [Ca2+]iresults from massive influx of Ca2+into the cytosol via
SK&F 96365–sensitive Ca2+channels that open in response to depletion of sarcoplasmic reticulum Ca2+stores. 5 The sustained or prolonged increase in [Ca2+]iis more complex. Because thapsigargin is an irreversible inhibitor of the sarcoplasmic reticulum Ca2+ATPase, the sarcoplasmic reticulum is incapable of refilling with Ca2+despite the increased availability of cytosolic free Ca2+. As a result, capacitative Ca2+entry across the sarcolemma is sustained, and a new steady state level of [Ca2+]iis achieved as other mechanisms regulating Ca2+extrusion (Na+–Ca2+exchanger and the sarcolemmal Ca2+ATPase) begin to offset the continued influx of Ca2+. If the sarcoplasmic reticulum is allowed to refill, as we have previously demonstrated using a reversible inhibitor of the sarcoplasmic reticulum Ca2+ATPase (cyclopiazonic acid), the response to capacitative Ca2+entry is only transient, and a sustained phase is not evident. 5

Thapsigargin-induced capacitative Ca2+entry does not involve activation of intracellular second messengers (e.g.
, 1,4,5 inositol triphosphate). The cellular mechanism that mediates capacitative Ca2+entry has been intensively investigated, although it has yet to be definitively identified. Various models have been postulated to explain how the sarcoplasmic reticulum communicates with the plasma membrane. These models can generally be divided into those that propose the existence of a diffusible factor and those that suggest that the signal is transferred via
protein phosphorylation and dephosphorylation. 2 Potentially important diffusible second messengers released from the storage organelles include cytochrome P450 metabolites, 20 G proteins, 21 or a low-molecular-weight compound called Ca2+influx factor. 22 Models based on phosphorylation and dephosphorylation interactions suggest that PKC activation either inhibits 14,23 or stimulates 24 capacitative Ca2+entry depending on the cell type, whereas tyrosine kinase or protein kinase A activation are consistently associated with activation of capacitative Ca2+entry. 14,25–27

Because capacitative Ca2+entry is involved in the regulation of [Ca2+]iand vasomotor tone in PASMCs, 5 the ion channel associated with capacitative Ca2+entry may serve as a cellular target for propofol. In cultured A10 and rat aortic smooth muscle cells, propofol was reported to inhibit voltage-gated Ca2+channels but to have no effect on capacitative Ca2+entry. 6 In contrast, propofol attenuated capacitative Ca2+entry in cultured aortic smooth muscle cells from normotensive rats, 28 and this effect was even more prominent in hypertensive rats. In our study using canine PASMCs, propofol caused a dose-dependent decrease in capacitative Ca2+entry. Typical free plasma concentrations of propofol range from 1 to 10 μm. Thus, propofol attenuated capacitative Ca2+entry at clinically relevant concentrations. Tyrosine kinase inhibition also attenuated capacitative Ca2+entry. However, in the presence of tyrosine kinase inhibition with tyrphostin 23, propofol further suppressed capacitative Ca2+entry. These results suggest that inhibition of tyrosine kinases is not the primary mechanism for propofol-induced inhibition of capacitative Ca2+entry. In contrast, inhibition of PKC with bisindolylmaleimide resulted in potentiation of capacitative Ca2+entry, whereas activation of PKC with phorbol 12-myristate 13-acetate resulted in attenuation of capacitative Ca2+entry. Moreover, pretreatment with bisindolylmaleimide prevented the propofol-induced attenuation of capacitative Ca2+entry. Taken together, these results suggest that propofol inhibits capacitative Ca2+entry via
a PKC-dependent mechanism. The cellular target for this propofol-induced, PKC-mediated attenuation of capacitative Ca2+entry remains to be elucidated. The propofol-induced attenuation of the peak response may either directly or indirectly be caused by PKC-dependent inhibition of Ca2+channels that mediate capacitative Ca2+entry, whereas attenuation of the sustained response may involve effects on the Na+–Ca2+exchanger or the sarcolemmal Ca2+ATPase. The current results are consistent with previous reports that propofol activates purified brain PKC. 29,30 Moreover, we have preliminary data that suggest that propofol increases myofilament Ca2+sensitivity in PASMCs via
a PKC-dependent mechanism. 31

In previous studies from our laboratory, 5,11 we demonstrated that α-adrenoreceptor–mediated Ca2+oscillations were not altered by the nonselective, broad-range protein kinase inhibitor staurosporine. In contrast, the Ca2+oscillations were abolished by SK&F 96365, an inhibitor of capacitative calcium entry, and were attenuated by tyrosine kinase inhibition with genestein or tyrphostin. In this study, selective inhibition of PKC potentiated capacitative Ca2+entry, whereas selective inhibition of tyrosine kinases attenuated capacitative Ca2+entry. Because staurosporine effectively blocks both tyrosine kinases and PKC, the lack of effect of staurosporine on the α-adrenoreceptor–mediated Ca2+oscillations is likely a result of offsetting effects on capacitative Ca2+entry, resulting in no net effect on the Ca2+oscillations. Although propofol (1–10 μm) attenuated capacitative Ca2+entry in this study, we previously reported that the propofol-induced attenuation of α-adrenoreceptor–mediated Ca2+oscillations was apparent only at supraclinical concentrations. 9 It should be noted that capacitative Ca2+entry is not the only mechanism regulating α-adrenoreceptor–mediated Ca2+oscillations. Moreover, regulation of capacitative Ca2+entry and Ca2+oscillations has not been entirely elucidated but appears to involve multiple mechanisms. In the current study, we demonstrated opposing actions of tyrosine kinases and PKC activation on capacitative Ca2+entry (CCE). It is possible that propofol alters multiple mechanisms involved in the regulation of the Ca2+oscillations, some of which may offset the effects of the other.

Although our results suggest that the effects of propofol on capacitative Ca2+entry are primarily mediated via
activation of PKC, alternative interpretations are possible. Propofol alone and tyrphostin alone inhibited capacitative Ca2+entry by approximately 30–35%. In the setting of tyrosine kinase inhibition, propofol further attenuated capacitative Ca2+entry by approximately 20%. Therefore, it could be argued that a portion of the inhibitory effect of propofol on capacitative Ca2+entry may be mediated via
inhibition of tyrosine kinases. However, this possibility seems unlikely because if the inhibitory effect of propofol on CCE is mediated by a pathway different from PKC, then propofol should continue to exert an inhibitory effect on CCE in the presence of PKC inhibition. Our results indicate that it did not. Given that PKC activation attenuated CCE and PKC inhibition abolished the propofol-induced attenuation in CCE, it seems reasonable to conclude that the effects of propofol are mediated (at least primarily) through PKC activation. This PKC-mediated attenuation of capacitative Ca2+entry induced by propofol does not appear to be a general characteristic of intravenous anesthetics. In preliminary studies, 32 thiopental had no effect on capacitative Ca2+entry, whereas ketamine attenuated capacitative Ca2+entry via
a mechanism that did not involve PKC. These results indicate that intravenous anesthetics can have differential effects on capacitative Ca2+entry that are mediated by more than one cellular mechanism.

In summary, propofol attenuates capacitative Ca2+entry in PASMCs. This effect is not altered by inhibition of tyrosine kinases but is abolished by inhibition of PKC. Capacitative Ca2+entry should be considered as a possible cellular target for anesthetic agents that alter vascular smooth muscle tone.